We have focused our studies in a number of different areas in FY2015: 1. For several years we have been interested in the role of cell surface and secreted TGF-beta in Treg Function. TGF-beta can have pleotropic effects on different cell types ranging from immune suppression, the promotion of fibrosis, to promotion/suppression of tumor growth. Activated regulatory T-cells (Tregs), but not activated T conventional cells, express the leucine rich repeat protein, GARP, which is responsible for surface localization of latent TGF-beta1. Although TGF-beta1 has been implicated in the suppressor function of Tregs, Treg conditional knock outs of TGF-beta1 or GARP display normal suppressor function in vitro. Cell surface associated TGF-beta1 on Treg cells may also mediates immunoregulatory functions. We have suggested an alternative role for Treg expressed cell surface TGF-beta1. Activation of co-cultures of activated Tregs and nave T cells resulted in induction of Foxp3 expression in a low percentage of the responder T cells in a TGF-beta1-dependent fashion. Other studies suggested that activated Treg cells could induce Th17 cells in the presence of IL-6. Although both the induction of Foxp3+ T cells and Th17 cells could have been mediated by TGF-beta1 that had been secreted by Tregs, we have demonstrated that the major source of the TGF-beta1 involved in both the induction of Foxp3+ T cells and IL-17-producing T cells is the GARP/LTGF-beta complex. Recently we and others have shown that release of active TGF-beta1 from the GARP/LTGF-beta1 complex was dependent on the expression integrin alphaVbeta8 on activated Treg Despite the conservation of GARP/LTGF-beta1 complex between mouse and man, very little is known about its functional role in vivo. The goal of the present studies was to define the role of the GARP/LTGF-beta1 complex in the biological functions of Treg. We have examined the in vivo Treg cell intrinsic functions of both GARP and TGF-beta1 as well as the cell extrinsic roles of GARP and TGF-beta1 in the induction of oral tolerance. We established that TGF-beta1 derived from the GARP/LTGF-beta1 complex modulates Treg cell quiescence/activation in vivo in the steady state and controls the expression of certain Treg cell membrane antigens (neuropilin-1, CD103) that play important roles in Treg cell function and tissue localization. Furthermore, we demonstrate the importance of Tregs for de novo establishment of oral tolerance via the GARP/LTGF-beta1 pathway. Lastly, we demonstrated that the intestinal microbiome also plays a critical role in the induction of oral tolerance by promoting Treg expansion and promoting a tolerance-inducing environment by enhancing the expression of the surface GARP/LTGF-beta1 complex on Treg cells in the mesenteric lymph node. 2. One of the critical issues regarding the potential therapeutic use of Tregs in vivo is the specificity of their inhibitory effects. To address this question, we generated allo-specific iTregs in vitro by stimulation of naive polyclonal Foxp3- T cells from Foxp3-GFP reporter mice with allogeneic DCs, TGFbeta1, and retinoic acid. The iTregs specifically suppressed responses to the inducing alloantigen in vitro, but had no effect on 3rd party stimulators. Similarly, when the allo-specific iTregs were transferred to immunocompetent recipients, they specifically suppressed responses induced by the inducing alloantigen, but not by 3rd party DC. To determine whether the allo-iTregs would suppress responses to foreign antigens driven by the target alloantigen presenting DC, we transferred allo-iTregs with antigen-specific T effector (Teff) cells specific for a foreign antigen presented by DCs exoressing the target alloantigen. When the alloantigen and the foreign antigen were present on the same DC, marked suppression of both the alloantigen and antigen responses were observed. However, when the alloantigen and foreign antigen were present on separate populations of DCs, only the responses to alloantigen were inhibited. We conclude that allo-specific iTreg represent a safe, promising strategy for the treatment of both GVHD and organ transplant rejection. We have extended these studies to determine the specificity of inhibition by antigen-specific Tregs. Tregs were generated in vitro from different strains of TCR transgenic mice. We first demonstrated that antigen-specific iTregs exhibited non-specific suppression in vitro. iTregs specific for one antigen could suppress responses to a distinct antigen either presented on the same APC or on separate APCs. Notably, different results were observed in vivo. iTreg specific for one antigen could only suppress immune responses to the same antigen, even when both antigenic peptides were present on the same APC. These results suggest that in vivo Tregs may not exhibit bystander suppression and that the mechanism of action of Tregs in this model involves direct targeting of MHC-antigenic peptide class II complexes rather than generalized deactivation of APC function. 3. Although we have primarily focused on the suppressive functions of Foxp3+ Treg, we have recently demonstrated that another population, gamma-delta T cells may exert potent suppression in an in vivo model of autoimmunity. Gamma-delta T cells have been shown to have immunoregulatory functions in several experimental autoimmune models. A mutation of the Foxp3 gene leads to the absence of regulatory T cells (Treg) and a fatal systemic autoimmune disease in scurfy mice. Transfer of scurfy lymphocytes to RAG deficient (-/-) recipients reproduces the inflammatory phenotype of the scurfy donor including hepatitis and pneumonitis. We have demonstrated that TCR alpha-/- recipients, that lack alpha-beta T cells, but have gamma-delta T cells and B cells, are significantly protected from the hepatitis and pneumonitis, but not the dermatitis, induced by adoptive transfer of scurfy lymphocytes. Co-transfer of gamma-delta T cells, but not B cells, prevented hepatitis and pneumonitis in RAG-/- recipients of scurfy lymphocytes. gamma-delta T cells in the TCRalpha-/- recipients of scurfy cells markedly expanded and expressed a highly activated (CD62LloCD44hi) phenotype. The activated gamma-delta T cells expressed high levels of CD39 and NKG2D on their cell surface. A high frequency of scurfy T cells in TCRalpha-/- recipients produced IL-10 suggesting that gamma-delta T cells may modulate cytokine production from scurfy T cells in TCRalpha-/- recipients. This study indicates that gamma-delta T cells may contribute to the maintenance of immunological homeostasis by suppressing autoreactive T cells in liver and lung.